Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro.

Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation (LLPS) in the absence of PrPSc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by ß-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid-solid phase transition could be the intermediate steps in the conversion of prions.

Electronic excitation spectra of jet-cooled phenyl isocyanate, m-tolyl isocyanate, and p-phenylene diisocyanate: Assignment of the cis and trans rotational isomers.

To assign cis and trans isomers of m-tolyl isocyanate (mTI) and p-phenylene diisocyanate (pPDI) in the electronic excitation transition, we measured the time-of-flight mass-selected resonant ionization spectra of jet-cooled phenyl isocyanate (PI), mTI, and pPDI in the region of the 275 nm first ππ* absorption system. In the excitation spectra of jet-cooled mTI and pPDI, cis- and trans-rotational isomers appeared as doublets. Isomers were assigned by analyzing the methyl-group internal rotation for mTI and by applying low-frequency bending vibrations to the mutual exclusion rule between the one- and two-photon spectra for pPDI. The electronic spectra of the three molecules observed in the jet were assigned to the transition to the first ππ* and third singlet excited states with the aid of time-dependent (TD)-B3LYP/aug-cc-pVDZ and TD-CAM-B3LYP/aug-cc-pVDZ calculations. The 0 - 0 band of PI was observed at 36 354 cm-1, those of the cis and trans isomers of mTI at 36 018 and 35 853 cm-1, respectively, and those of the cis and trans isomers of pPDI at 34 437 and 34 383 cm-1, respectively. All vibronic bands were diffuse, probably because of internal conversion to two singlet nπ* states. For mTI, based on changes in the barrier height of methyl-group internal rotation upon excitation, the Hammett-σm of PI was determined to be -0.12.

Targeted Delivery of Miconazole Employing LL37 Fragment Mutant Peptide CKR12-Poly (Lactic-Co-Glycolic) Acid Polymeric Micelles.

We previously reported that conjugates of antimicrobial peptide fragment analogues and poly (lactic-co-glycolic) acid (PLGA) enhance antimicrobial activity and that the conjugated micelle structure is an effective tool for antimicrobial drug delivery. In recent years, the delivery of antimicrobial peptides to targets for antimicrobial activity has attracted attention. In this study, we targeted Candida albicans, a causative organism of catheter-related bloodstream infections, which is refractory to antimicrobial agents and is currently a problem in medical practice. We evaluated the antifungal activity of CKR12 (a mutant fragment of the human cathelicidin peptide, LL-37)-PLGA-miconazole (MCZ) micelles using nanotechnology with MCZ delivery. The prepared CKR12-PLGA-MCZ micelles were characterised by measuring dynamic light scattering, zeta potential, dilution stability, and drug release. CKR12-PLGA-MCZ micelles showed higher antifungal activity than CKR12-PLGA micelles and MCZ solution. Furthermore, scanning and transmission electron microscopy suggested that CKR12-PLGA-MCZ micelles disrupted both cell wall and cell membrane of C. albicans. Our results revealed a synergistic effect of antifungal activity using a combination of antimicrobial peptide fragment analogues and MCZ, and that MCZ is a promising tool for the delivery to target microorganisms.

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

Prion protein interacts with the metabotropic glutamate receptor 1 and regulates the organization of Ca2+ signaling.

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca2+ signaling. In this study, we evaluated the binding of PrP to metabotropic glutamate receptor 1 (mGluR1) and found that wild-type PrP (PrP-wt) and mGluR1 co-immunoprecipitated in dual-transfected Neuro-2a (N2a) cells. Fluorescence resonance energy transfer analysis revealed an energy transfer between mGluR1-Cerulean and PrP-Venus. In order to determine whether PrP can modulate mGluR1 signaling, we performed Ca2+ imaging analyses following repetitive exposure to an mGluR1 agonist. Agonist stimulation induced synchronized Ca2+ oscillations in cells coexpressing PrP-wt and mGluR1. In contrast, N2a cells expressing PrP-ΔN failed to show ligand-dependent regulation of mGluR1-Ca2+ signaling, indicating that PrP can bind to mGluR1 and modulate its function to prevent irregular Ca2+ signaling and that its N-terminal region functions as a molecular switch during Ca2+ signaling.

Generation, optimization and characterization of novel anti-prion compounds.

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.

Type I interferon protects neurons from prions in in vivo models.

Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

Postmortem Quantitative Analysis of Prion Seeding Activity in the Digestive System.

Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob disease, recent reports suggest that the seeding activity of Creutzfeldt-Jakob disease prions accumulates in various non-neuronal organs including the liver, kidney, and skin. Therefore, we reanalyzed autopsy samples collected from patients with sporadic and genetic human prion diseases and found that seeding activity exists in almost all digestive organs. Unexpectedly, activity in the esophagus reached a level of prion seeding activity close to that in the central nervous system in some CJD patients, indicating that the safety of endoscopic examinations should be reconsidered.

Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion.

The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrP(Sc)). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrP(Sc). To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to ß-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fib(Ch)) and 22L-seeded (1st-rPrP-fib(22L)) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the ß-sheets resembled those of the original PrP(Sc). Furthermore, the conformational stability of 1st-rPrP-fib(Ch) was significantly higher than that of 1st-rPrP-fib(22L), as with Chandler and 22L PrP(Sc). The survival periods of mice inoculated with 1st-rPrP-fib(Ch) or 1st-rPrP-fib(22L) were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation. Importance: The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrP(Sc)). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrP(Sc) formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.

Biweekly THP-COP therapy for newly diagnosed peripheral T-cell lymphoma patients.

Pirarubicin tetrahydropyranyl adriamycin (THP-ADR) is an analogue of doxorubicin. This agent exhibits activity against some doxorubicin-resistant cell lines. We performed a phase II study of biweekly THP-COP [50 mg/m(2) pirarubicin, 750 mg/m(2) cyclophosphamide, 1.4 mg/m(2) vincristine (2.0 mg maximum) on day 1, and 100 mg/body predonisolone on days 1-5] in patients with peripheral T-cell lymphoma (PTCL). Seventeen patients with newly diagnosed PTCL were enrolled. Histological diagnoses were of PTCL, not otherwise specified (n = 5), or angioimmunoblastic T-cell lymphoma (n = 12). All diagnostic specimens including those of the historical control group were centrally reviewed by hematological pathologists. All patients received six cycles of biweekly THP-COP. The patient group included 13 male and 4 female patients, with a median age of 62 years. The median follow-up time in surviving patients was 30 months. Overall response rate was 94% with 15 cases of complete remission (88%). The 3-year progression-free survival and overall survival rates were 57% and 75%, respectively. The most frequent adverse events associated with biweekly THP-COP were leukocytopenia (100%), neutropenia (100%), and lymphopenia (100%), followed by alopecia (92%) and anaemia (88%). All of these occurred only transiently, and the patients subsequently recovered. Biweekly THP-COP is a safe and promising therapy for patients with newly diagnosed PTCL. This study is registered in a public database (UMIN000010485).

European Treatment and Outcome Study score does not predict imatinib treatment response and outcome in chronic myeloid leukemia patients.

The Sokal and Hasford scores were developed in the chemotherapy and interferon era and are widely used as prognostic indicators in patients with chronic myeloid leukemia (CML). Recently, a new European Treatment and Outcome Study (EUTOS) scoring system was developed. We performed a multicenter retrospective study to validate the effectiveness of each of the three scoring systems. The study cohort included 145 patients diagnosed with CML in chronic phase who were treated with imatinib. In the EUTOS low- and high-risk groups, the cumulative incidence of complete cytogenetic response (CCyR) at 18 months was 86.9% and 87.5% (P = 0.797) and the 5-year overall survival rate was 92.6% and 93.3% (P = 0.871), respectively. The cumulative incidence of CCyR at 12 months, 5-year event-free survival and 5-year progression-free survival were not predicted using the EUTOS scoring system. However, there were significant differences in both the Sokal score and Hasford score risk groups. In our retrospective validation study, the EUTOS score did not predict the prognosis of patients with CML in chronic phase treated with imatinib.

Peripheral blood absolute lymphocyte/monocyte ratio as a useful prognostic factor in diffuse large B-cell lymphoma in the rituximab era.

OBJECTIVES: The tumor microenvironment, including tumor-infiltrating lymphocytes and myeloid-derived cells, is an important factor in the pathogenesis and clinical behavior of malignant lymphoma. However, the prognostic significance of peripheral lymphocytes and monocytes in lymphoma remains unclear. METHODS: We evaluated the prognostic impact of the absolute lymphocyte count (ALC), absolute monocyte count (AMC), and lymphocyte/monocyte ratio (LMR) in 359 diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: The median follow-up time of the surviving patients was 58 months. Low ALC and an elevated AMC were both associated with poor survival rates. Receiver operating characteristic curve analysis showed that LMR was the best predictor of survival, with 4.0 as the cutoff point. Patients with LMR ≤4.0 were more likely to have an aggressive tumor, and this was associated with poor treatment responses. Patients with LMR ≤4.0 at diagnosis had significantly poorer overall survival (OS) and progression-free survival (PFS) than those with LMR >4.0. Multivariate analysis, which included prognostic factors of the International Prognostic Index, showed LMR ≤4.0 to be an independent predictor for the OS (hazard ratio [HR], 2.507; 95% confidence interval [CI], 1.255-5.007; P = 0.009) and PFS (HR, 2.063; 95% CI, 1.249-3.408; P = 0.005). CONCLUSIONS: The LMR at diagnosis, as a simple index which reflects host systemic immunity, predicts clinical outcomes in DLBCL patients treated with R-CHOP.

Direct evidence of generation and accumulation of ß-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for ß-form prion protein.

We prepared ß-sheet-rich recombinant full-length prion protein (ß-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this ß-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to ß-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of ß-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of ß-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing ß-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to ß-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

Protective role of interferon regulatory factor 3-mediated signaling against prion infection.

Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP(Sc) was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP(Sc) formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.

RCAS1 increases cell morphological changes in murine fibroblasts by reducing p38 phosphorylation.

Receptor­binding cancer antigen expressed on SiSo cells (RCAS1) is a tumor­associated antigen that is expressed in a number of human malignancies. RCAS1 acts as a ligand for a putative RCAS1 receptor that is present on various human cells including T and B lymphocytes and natural killer cells, in which it induces cell growth inhibition and apoptosis. It has been suggested that RCAS1 might serve an important role in tumor cell evasion from the host immune system. In fact, RCAS1 expression is related to malignant characteristics including tumor size, invasion depth, clinical stage and poor overall survival. The authors previously established doxycycline­induced RCAS1 overexpression murine fibroblast L cells to analyze the biological functions of RCAS1 and reported that its expression inhibited cell cycle progression via the downregulation of cyclin D3, which subsequently induced apoptosis. Additionally, it was found that RCAS1 expression induced cell morphological changes prior to caspase­mediated apoptosis. Thus, the present study examined signaling pathways associated with changes in cell morphology that were induced by RCAS1 expression. The data showed that increased RCAS1 expression caused a reduction in actin stress fibers and decreased cofilin phosphorylation. Recent studies have shown that p38 signaling regulates actin polymerization. The data the present study showed that increased RCAS1 expression significantly decreased p38 phosphorylation.

Development of an Organ-Directed Exosome-Based siRNA-Carrier Derived from Autologous Serum for Lung Metastases and Testing in the B16/BL6 Spontaneous Lung Metastasis Model.

Exosomes are nano-sized extracellular vesicles that are known to carry various messages to distant cells. It was recently reported that cancer-derived exosomes are orientated to metastatic organs. However, there are no reports on drug carrier development using autologous serum-derived exosomes in vivo. The purpose of this study was to deliver therapeutic siRNAs for melanoma lung metastases using autologous serum-derived exosomes. Primary tumors were induced by subcutaneously injecting melanoma cells into the hindlimbs of female C57BL/6 mice. Primary tumors were surgically removed on day 14. On day 21 after tumor removal, lung metastases were evaluated. Exosomes were isolated from serum collected from mice on days 0, 3, 7, 10, and 14 after primary tumor inoculation. After isolating serum exosomes, siRNA-loaded exosomes were prepared. siRNA-loaded exosomes were intravenously injected into the B16/BL6 spontaneous lung metastasis model mice on days 0, 3, 7, and 10 after tumor removal. siRNA-loaded exosomes prepared with autologous serum-derived exosomes significantly decreased the number of metastatic lung colonies. Autologous serum-derived exosomes, which have high organ accumulation, could potentially be used as efficient carriers of therapeutic siRNAs for melanoma patients with lung metastases.

Preparation of siRNA-PLGA/Fab'-PLGA mixed micellar system with target cell-specific recognition.

Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(D,L-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA-PLGA/Fab'-PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA-PLGA/Fab'-PLGA mixed micelles were added to cells compared with siRNA-PLGA micelles. siRNA-PLGA/Fab'-PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA-PLGA/Fab'-PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer.

Enhancing the anticancer efficacy of a LL-37 peptide fragment analog using peptide-linked PLGA conjugate micelles in tumor cells.

LL-37, a well-known antimicrobial human peptide, is a cationic peptide that provides an important antimicrobial defense mechanism in damaged skin. Accumulating evidence indicates that LL-37 also displays an anticancer effect in colon cancer, gastric cancer, hematologic malignancy and oral squamous cell carcinoma. However, anticancer activity of LL-37 peptide fragment analogs has not been reported. Poor intercellular translocation may be one of the causes for this lack of observed anticancer activity. In this study, a LL-37 peptide fragment analog with cysteine at the N-terminus was conjugated with the biodegradable polymer, lactic acid/glycolic acid copolymer (PLGA), using the thiol group of cysteine. The purpose of this study was to improve the cell permeability of the peptide using a micellar system and then evaluate the anticancer activity. Cell proliferation, migration, and invasion assays were performed to evaluate the anticancer activity in four cancer cell lines with high metastasis, HM-1, B16/BL6, HeLa, and HepG2. The LL-37 fragment peptide analog-linked PLGA conjugate was shown to effectively inhibit cell proliferation, migration, and invasion and had increased cell permeability in all the cancer cell lines, compared with the peptide alone. These results suggested that LL-37 fragment peptide analog (CKR12)-linked PLGA conjugate micelles could be useful in the development of cancer therapeutics.

Dextran sulphate inhibits an association of prions with plasma membrane at the early phase of infection.

The defining characteristic of prion diseases is conversion of a cellular prion protein (PrPC) to an abnormal prion protein (PrPSc). The exogenous attachment of PrPSc to the surface of a target cell is critical for infection. However, the initial interaction of PrPSc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrPSc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22 L-infected brain homogenates and revealed that PrPSc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrPC-deficient hippocampus cell lines. We also demonstrate here that PrPSc from 22 L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrPSc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.